1. Field of the Invention
The present invention relates to medically useful preparations based on hyaluronic acid, a naturally-occurring substance found in animal tissue, and especially in rooster comb, vitreous humour, umbilical cords, and synovial fluid of mammals. More particularly, the present invention relates to injectable formulations containing a fraction of hyaluronic acid, which is pyrogen-free, heat stable and has been purified and heat-sterilized. Specifically, the present invention is directed to a heat stable, purified, pyrogen-free, heat-sterilized fraction of hyaluronic acid, formulations and compositions containing such fraction of hyaluronic acid suitable for intra-articular treatment of animals, methods of preparing a formulation containing such fraction of hyaluronic acid suitable for this purpose, and methods for intra-articular treatment of animals with formulations and compositions containing such fraction of hyaluronic acid.
2. Description of Background and Material Information
It is known in the art that hyaluronic acid (HA) has the potential to be a valuable therapeutic agent in the treatment of joint disorders. Introduction of HA into a diseased or malfunctioning joint has the potential to prevent further deterioration of joint function, and even to restore the joint to its normal, healthy condition. Unfortunately, the injection of prior art HA preparations into joint synovial spaces results in serious side-effects in a significant number of cases. The principal side-effects are pain and inflamation of the joint, often impairing normal activity for weeks or months following treatment. Repeated injections of prior art HA preparations may even cause sensitization in the subject, causing progressively more severe side-effects.
Hyaluronic acid (HA) occurs naturally in joint synovial fluid, where it plays a lubricating role, and may have biological activity as well. HA is a mucopolysaccharide, and may alternatively be referred to as a glycosaminoglycan. The repeating unit of the hyaluronic acid molecule is a disaccharide consisting of D-glucuronic acid and N-acetyl-D-glucosamine. Because hyaluronic acid possesses a negative charge at neutral pH, it is soluble in water, where it forms highly viscous solutions. The D-glucuronic acid unit and N-acetyl-D-glucosamine unit are bonded through a glycosidic, beta(1-3) linkage, while each disaccharide unit is bonded to the next disaccharide unit through a beta(1-4) linkage. The beta(1-4) linkages may be broken through hydrolysis with the enzyme hyaluronidase.
A variety of substances, commonly referred to hyaluronic acid, have been isolated by numerous methods from various tissue sources including umbilical cords, skin, vitreous humour, synovial fluid, tumors, haemolytic streptococci pigskin, rooster combs, and the walls of veins and arteries.
Conventional methods for obtaining hyaluronic acid result with a product having differing properties and a wide range of viscosities. U.S. Pat. No. 2,585,546, HADIAN, is an example of a method for obtaining hyaluronic acid which involves extracting acetone-washed umbilical cords with a dilute salt solution, acidifying the resulting extract, removing the clot so formed, precipitating some hyaluronic acid with protein from the acidified extract with ammonium sulfate, agitating the liquid with pyridine, precipitating another fraction highly contaminated with protein, followed by more ammonium sulfate which forces some pyridine out of solution along with the high viscosity hyaluronic acid. The hyaluronic acid collects at the interface between the two liquid phases and may be separated by filtration, centrifugation or other usual procedure. A modification of this process involves the fractionation of the acidic salt extract from umbilical cords with alcohol and ammonium sulfate. Alcohol is added to the acidic salt extract, and the resulting precipitate is removed. Solid ammonium sulfate is added to the liquid until saturation and the solution forms two phases with a precipitate of hyaluronic acid at the interface.
U.S. Pat. No. 3,396,081, BILLEK, produces a dry powder of hyaluronic acid obtained from a source of hyaluronic acid, such as animal organs including the vitreous body of the eye, umbilical cords, and the like, or from bacterial cultures producing hyaluronic acid. A suspension of the resultant dry powder is heated in water for a short time in an alkaline range whereby the protein content is denatured. After adjustment of the optimum pH and temperature range for the enzyme used, the protein is decomposed by proteolytic ferments, preferably by a hydrolase mixture of Aspergillus oryzae. After removing the free amino acids and mineral salts by treatment with ion exchanges, an impure hyaluronic acid solution still containing protein is obtained. The resultant solution of impure hyaluronic acid containing residual protein is then adjusted to an acid pH of about 3-4 in which the impurities form with hyaluronic acid an insoluble complex, while part of the hyaluronic acid itself functions as precipitate for the impurities, particularly for the residual proteins which can otherwise be removed only with difficulty without depolymerization of the hyaluronic acid, whereupon the resulting insoluble complex compounds are separated by high speed centrifuging. After a sodium salt, the resultant hyaluronic acid solution with a concentration of 0.2% in water has a relative specific viscosity of 20 and constitutes a water-clear solution disclosure as being free from proteins, antigens, and pyrogens, which is disclosed as being suitable for heat sterilization without experiencing a considerable drop in viscosity.
U.S. Pat. No. 3,862,003, OKUYAMA et al., is directed to a method of extracting mucopolysaccharides from connective tissues of animals, which involves exposing the connective tissue with water at a temperature of 105.degree.-150.degree. C. under an elevated pressure, subjecting the resultant extract to a protease treatment and/or alkali treatment, and then separating and recovering mucopolysaccharides.
U.S. Pat. No. 4,141,973, BALAZS, is directed to the production of an ultra-pure, high molecular weight hyaluronic acid fraction which is obtained from animal tissue containing hyaluronic acid by a process which involves moving blood from the animal tissue containing hyaluronic acid, extracting hyaluronic acid from the blood, deproteinizing the hyaluronic acid extract, and removing any unidentified inflammation causing agents present therein by treating the deproteinized hyaluronic acid extract at a pH of 6.0-7.0 with a volume of chloroform at least about equal to that of the deproteinized extract, to form a two-phase mixture which is then stirred, sufficiently to insure intimate contact with the two phases at about 15.degree.-40.degree. C., followed by separating out and discarding the chloroform phase.
U.S. Pat. No. 4,517,296, BRACKE et al., is directed to the preparation of hyaluronic acid in high yield from streptococcus bacteria by fermenting the bacteria under anerobic conditions in a CO.sub.2 enriched growth medium, separating the bacteria from the resulting broth and isolating the hyaluronic acid from the remaining constituents of the broth. Separation of the microorganisms from the hyaluronic acid is facilitated by killing the bacteria with trichloroacetic acid. After removal of the bacteria cells and concentration of the higher molecular weight fermentation products, the hyaluronic acid is isolated and purified by precipitation, resuspension and reprecipitation.
Despite such prior arts attempts in the preparation of hyaluronic acid, conventional procedures for doing so have been hampered by the need to balance inherently conflicting objectives, i.e., the elimination of inflammatory or pyrogenic properties by extensive purification, and maintaining a high viscosity of the preparation, which generally decreases in response to each successive isolation or purification step. Resolution of this dilemma has been complicated by the fact that the agents responsible for the inflamatory and/or pyrogenic side-effects of the prior art preparations are not well understood, making their efficient elimination extremely difficult.
The present invention overcomes the difficulties of the prior art through the discovery of a hyaluronic acid preparation which contains HA fractions which are heat stable, purified, pyrogen-free and heat-sterilized. Although the HA fractions may have a relatively low molecular weight, the HA fractions are nevertheless highly effective in the treatment of joint disorders while avoiding the side-effects which are a major drawback of the prior art preparations.